L1/2 - Influenza Flashcards
Which influenza viruses have 8 segments?
A
B
Which influenza viruses have 7 segments
C
D
What are the 3 membrane proteins of influenza?
NA
HA
M2
Why do the segments not look like linear strands of DNA?
segments are wrapped up in RNA interactions and RNA-protein interactions
RNA-PROTEIN COMPLEXES
What are the two interactions that hold the RNA segment ends together
Watson-Crick interactions
PAN HANDLE STRUCTURE
5’ and 3’ ends interacts with RdRp
What protein does the RNA molecule interacts with?
NP - entirely covers
What 2 properties of NP
RNA binding via pos-charged residues in groove
NP-NP interaction via tail loop
What kind of conformation do influenza RNPs adopt?
hairpin
double helical conformation
antiparallel
held by NP-NP interactions
What 3 segments make the RdRp
PB1
PB2
PA
How can the 8 segments of influenza achieve more coding potential?
splicing
What is the 1st stage of the influenza virus life cycle?
virus attachment
How does virus attachment take place in influenza
mediated by HA
sialic acid recognised
target epithelial cells
virus internalised within endosome
different types of sialic acid linkage
Why does the Human and avian flu have different sialic acid linkages?
HA proteins show specificity to just one SA type
What sialic acid linkages does human influenza infect?
a2-6
What sialic acid linkages does avian influenza infect?
a2-3
How can specificity of sialic acid binding be switched?
single residue changes
e.g. Glu90
How does flu fuse with endosomes
pH 5 - mediates conformational shift in the HA protein
What is stage 2 of the influenza life cycle?
virus entry
How does the influenza virus escape the endosome?
fusion of viral & endosomal membranes
RNP segments released
mediated by HA protein and fusion peptide
How does the fusion peptide allow fusion to occur?
fusion peptide buried in HA0
HA1/HA2 cleavage = repositioning at amino-terminus
sticks into endosomal membrane
What is the conformation change that happens to the fusion peptide?
2 helices in HA2 align
flips fusion peptide upwards
insertion
what are the 2 CRITICAL FUNCTIONS of the HA protein?
1) virus attachment via sialic acid
2) virus entry by fusion of viral & endosomal membranes
What is the 3rd stage of the influenza life cycle?
RNP release
What happens during RNP release from matrix proteins?
mediated by M2
M2 x 4 = tetramer
M2 - influx of H+ ions into virion
What is the 4th stage of the influenza life cycle?
RNP import
What happens during RNP import
move from cytoplasm to nucleus
individual NP monomers - 2 import signals
what is the ONLY NLS exposed on formed RNPs?
NLS1
recruits importin alpha
recruits importin b
RNP/importin complex binds pore and enters
What is the 5th stage of the influenza life cycle?
RNA synthesis
what are the 2 processes of RNA synthesis?
Transcription
Replication
What is the polymerase structure for influenza?
3 subunits
4 holes lead to centre of the polymerase
What happens during influenza transcription
results in 1 RNA transcript PER SEGMENT
resemble cellular mRNAs
What are the 4 stage of influenza transcription?
RdRp PB2 binds cellular mRNA in nucleus - PA steals 5’ cap
RdRP + capped oligonucleotide bind to negative RNA genome
RdRP PB1 - use capped RNA, transcription
RdRp - move along templated, PB1 generates RNA transcript with polyA tail & 5’ cap
(NOT ENCAPSIDATED)
What is the fate of influenza mRNA?
some stay in cytoplasm
some exported through nuclear pores - translated to viral proteins
How are some influenza proteins imported back into the nucleus?
imported by NLS
How can NS2 protein get back inside with nucleus without NLS?
it is an export signal - cannot have import
very small
What are the 4 stages of influenza replication
RdRP binds 3’ end of RNA segment (no primer)
RdRp travels along segment - 3’ to 5’ direction - complimentary copy
NP wraps RNA = RNPs
cycles = amplification of genomes
Why does transcription have to occur before replication in influenza?
requirement for NP
What is the 6th stage of the influenza life cycle?
RNP export
What is involved in RNP export (influenza)?
binding of M1 and NS2
- RNPs associated with M1 in nucleus
- NS2 binding M forming outer layer on RNP
- NS2 has export signal, dominant over other signal - NS2 covers NLS
- CRM1 binds to NS2 and helps export
What protein, along with NS2, mediates RNP export in influenza?
CRM1
binds Ran-GTP
exits through NPC
Where must RNPs reach?
the apical surface - with the 3 envelope proteins
How do RNPs traffic by recycling endosomes?
interactions of RNPs with Rab11
M1 associates with cytoplasmic tails of the 3 envelope proteins
What is another name for M1?
MASTER RECRUITER
Why is viral release polarised towards apical surface and not the basolateral surface?
recycling endosomes only move to the apical surface
What is the 7th stage of the influenza life cycle?
virus assembly
What happens during virus assembly of influenza?
segments are selected
7+1 arrangement
What is evidence for direct RNP interactions in virions between segments?
segments interact via RNA-RNA interactions
multiple interactions allow for sequence variations without impaired packaging
e.g. PB1-NP interaction
What is the 8the stage of the influenza virus life cycle?
virus release
What happens during influenza virus release?
NA solves the issue of binding on the sialic acid of the old cell
NA cleaves sialic acid from previously infected cell - only new ones infected
